Efficacy of IFN-γ, sCD40L, and Poly(I:C) Treated Bone Marrow-Derived Macrophages in Murine Mammary Carcinoma.

INTRODUCTION: Here, we explored methods to generate anti-tumor bone marrow-derived macrophages (BMDM) and how delivery of the BMDM at early tumor sites could impact disease progression. METHODS: BMDM treated with IFN-γ, sCD40L, poly(I:C), and a combination of the three were assessed. RESULTS: Treatment with sCD40L had no significant impact on the BMDM. Treating BMDM with IFN-γ impacted IL-1ß, MHC Class II, and CD80 expression. While poly(I:C) treatment had a greater impact on the BMDM than IFN-γ when assessed by the in vitro assays, the BMDM treated with poly (I:C) had mixed results in vivo where they decreased growth of the EMT6 tumor, did not impact growth of the 168 tumor, and enhanced growth of the 4T1 tumor. The combination of poly(I:C), IFN-γ, and sCD40L had the greatest impact on the BMDM in vitro and in vivo. Treatment with all three agonists resulted in increased IL-1ß, TNF-α, and IL-12 expression, decreased expression of arginase and mrc, increased phagocytic activity, nitrite production, and MHC Class II and CD80 expression, and significantly impacted growth of the EMT6 and 168 murine mammary carcinoma models. DISCUSSION: Collectively, these data show that treating BMDM with poly(I:C), IFN-γ, and sCD40L generates BMDM with more consistent anti-tumor activity than BMDM generated with the individual agonists.

LYVE-1-expressing Macrophages Modulate the Hyaluronan-containing Extracellular Matrix in the Mammary Stroma and Contribute to Mammary Tumor Growth.

Macrophages represent a heterogeneous myeloid population with diverse functions in normal tissues and tumors. While macrophages expressing the cell surface marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) have been identified in stromal regions of the normal mammary gland and in the peritumoral stroma, their functions within these regions are not well understood. Using a genetic mouse model of LYVE-1+ macrophage depletion, we demonstrate that loss of LYVE-1+ macrophages is associated with altered extracellular matrix remodeling in the normal mammary gland and reduced mammary tumor growth in vivo. In further studies focused on investigating the functions of LYVE-1+ macrophages in the tumor microenvironment, we demonstrate that LYVE-1 expression correlates with an increased ability of macrophages to bind, internalize, and degrade hyaluronan. Consistent with this, we show that depletion of LYVE-1+ macrophages correlates with increased hyaluronan accumulation in both the normal mammary gland and in mammary tumors. Analysis of single-cell RNA sequencing of macrophages isolated from these tumors reveals that depletion of LYVE-1+ macrophages in tumors drives a shift in the majority of the remaining macrophages toward a proinflammatory phenotype, as well as an increase in CD8+ T-cell infiltration. Together, these findings indicate that LYVE-1+ macrophages represent a tumor-promoting anti-inflammatory subset of macrophages that contributes to hyaluronan remodeling in the tumor microenvironment. SIGNIFICANCE: We have identified a macrophage subset in mouse mammary tumors associated with tumor structural components. When this macrophage subset is absent in tumors, we report a delay in tumor growth and an increase in antitumor immune cells. Understanding the functions of distinct macrophage subsets may allow for improved therapeutic strategies for patients with breast cancer.

Chemoprotective and immunomodulatory potential of Lactobacillus reuteri against cadmium chloride-induced breast cancer in mice.

INTRODUCTION: The current study aimed to investigate the role of probiotic Lactobacillus reuteri for the treatment and prevention of breast cancer. MATERIALS AND METHODS: Breast cancer was induced by using Cadmium Chloride (Cd) (2 mg/kg) in group II. Tamoxifen was administered to group III. Group IV was treated with Lactobacillus reuteri. Group V was treated with Cd for one month and divided into three subgroups including VA, VB, and VC which were treated with tamoxifen, Lactobacillus reuteri, and tamoxifen + Lactobacillus reuteri, respectively. RESULTS: Significantly higher levels of TNF-α (40.9 ± 4.2 pg/mL), IL-6 (28.0 ± 1.5 pg/mL), IL-10 (60.2 ± 2.0 pg/mL), IFN-γ (60.2 ± 2.0 pg/mL), ALAT (167.2 ± 6.2 U/l), ASAT (451.6 ± 13.9 U/l), and MDA (553.8 ± 19.6 U/l) was observed in Cd group. In comparison, significantly lower levels of TNF-α (18.0 ± 1.1 pg/mL), IL-6 (9.4 ± 0.4 pg/mL), IL-10 (20.8 ± 1.1 pg/mL), IFN-γ (20.8 ± 1.1 pg/mL), ALAT (85.2 ± 3.6 U/l), ASAT (185 ± 6.9 U/l), and MDA (246.0 ± 7.5 U/l) were observed in group Cd + Tam + LR. Liver histopathology of the Cd group showed hemorrhage and ductal aberrations. However, mild inflammation and healthier branched ducts were observed in treatment groups. Furthermore, the renal control group showed normal glomerular tufts, chronic inflammation from the Cd group, and relatively healthier glomerulus with mild inflammation in treatment groups. CONCLUSION: Hence, the preventive and anticancerous role of probiotic Lactobacillus reuteri is endorsed by the findings of the current study.

PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

PD-1 monoclonal antibodies enhance the cryoablation-induced antitumor immune response: a breast cancer murine model research.

BACKGROUND: It has been demonstrated that cryoablation (Cryo) causes specific T-cell immune responses in the body; however, it is not sufficient to prevent tumor recurrence and metastasis. In this report, we evaluated changes in the tumor immune microenvironment (TIME) in distant tumor tissues after Cryo and investigated the immunosuppressive mechanisms that limit the efficacy of Cryo. METHODS: Bilateral mammary tumor models were established in mice, and we first observed the dynamic changes in immune cells and cytokines at different time points after Cryo. Then, we confirmed that the upregulation of PD-1 and PD-L1 signaling in the contralateral tumor tissue was closely related to the immunosuppressive state in the TIME at the later stage after Cryo. Finally, we also evaluated the synergistic antitumor effects of Cryo combined with PD-1 monoclonal antibody (mAb) in the treatment of breast cancer (BC) mouse. RESULTS: We found that Cryo can stimulate the body's immune response, but it also induces immunosuppression. The elevated PD-1/PD-L1 expression in distant tumor tissues at the later stage after Cryo was closely related to the immunosuppressive state in the TIME but also created the conditions for Cryo combined with PD-1 mAb for BC mouse treatment. Cryo + PD-1 mAb could improve the immunosuppressive state of tumors and enhance the Cryo-induced immune response, thus exerting a synergistic antitumor effect. CONCLUSIONS: The PD-1/PD-L1 axis plays an important role in suppressing Cryo-induced antitumor immune responses. This study provides a theoretical basis for Cryo combined with PD-1 mAb therapy in clinical BC patients.

Antigen presentation and interferon signatures in B cells driven by localized ablative cancer immunotherapy correlate with extended survival.

Rationale: B cells have emerged as key regulators in protective cancer immunity. However, the activation pathways induced in B cells during effective immunotherapy are not well understood. Methods: We used a novel localized ablative immunotherapy (LAIT), combining photothermal therapy (PTT) with intra-tumor delivery of the immunostimulant N-dihydrogalactochitosan (GC), to treat mice bearing mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT). We used single-cell RNA sequencing to compare the transcriptional changes induced by PTT, GC and PTT+GC in B cells within the tumor microenvironment (TME). Results: LAIT significantly increased survival in the tumor-bearing mice, compared to the treatment by PTT and GC alone. We found that PTT, GC and PTT+GC increased the proportion of tumor-infiltrating B cells and induced gene expression signatures associated with B cell activation. Both GC and PTT+GC elevated gene expression associated with antigen presentation, whereas GC elevated transcripts that regulate B cell activation and GTPase function and PTT+GC induced interferon response genes. Trajectory analysis, where B cells were organized according to pseudotime progression, revealed that both GC and PTT+GC induced the differentiation of B cells from a resting state towards an effector phenotype. The analyses confirmed upregulated interferon signatures in the differentiated tumor-infiltrating B cells following treatment by PTT+GC but not by GC. We also observed that breast cancer patients had significantly longer survival time if they had elevated expression of genes in B cells that were induced by PTT+GC therapy in the mouse tumors. Conclusion: Our findings show that the combination of local ablation and local application of immunostimulant initiates the activation of interferon signatures and antigen-presentation in B cells which is associated with positive clinical outcomes for breast cancer. These findings broaden our understanding of LAIT's regulatory roles in remodeling TME and shed light on the potentials of B cell activation in clinical applications.

T Cells Promote Metastasis by Regulating Extracellular Matrix Remodeling following Chemotherapy.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. SIGNIFICANCE: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197.

Parity-induced changes to mammary epithelial cells control NKT cell expansion and mammary oncogenesis.

Pregnancy reprograms mammary epithelial cells (MECs) to control their responses to pregnancy hormone re-exposure and carcinoma progression. However, the influence of pregnancy on the mammary microenvironment is less clear. Here, we used single-cell RNA sequencing to profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis indicates an expansion of γδ natural killer T-like immune cells (NKTs) following pregnancy and upregulation of immune signaling molecules in post-pregnancy MECs. We show that expansion of NKTs following pregnancy is due to elevated expression of the antigen-presenting molecule CD1d on MECs. Loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKTs, results in mammary oncogenesis. Collectively, our findings illustrate how pregnancy-induced changes modulate the communication between MECs and the immune microenvironment and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

Immunoprotective effect of an in silico designed multiepitope cancer vaccine with BORIS cancer-testis antigen target in a murine mammary carcinoma model.

In our previous study, immunoinformatic tools were used to design a novel multiepitope cancer vaccine based on the most immunodominant regions of BORIS cancer-testis antigen. The final vaccine construct was an immunogenic, non-allergenic, and stable protein consisted of multiple cytotoxic T lymphocytes epitopes, IFN-γ inducing epitopes, and B cell epitopes according to bioinformatic analyzes. Herein, the DNA sequence of the final vaccine construct was placed into the pcDNA3.1 vector as a DNA vaccine (pcDNA3.1-VAC). Also, the recombinant multiepitope peptide vaccine (MPV) was produced by a transfected BL21 E. coli strain using a recombinant pET-28a vector and then, purified and screened by Fast protein liquid chromatography technique (FPLC) and Western blot, respectively. The anti-tumor effects of prophylactic co-immunization with these DNA and protein cancer vaccines were evaluated in the metastatic non-immunogenic 4T1 mammary carcinoma in BALB/c mice. Co-immunization with the pcDNA3.1-VAC and MPV significantly (P < 0.001) increased the serum levels of the MPV-specific IgG total, IgG2a, and IgG1. The splenocytes of co-immunized mice exhibited a significantly higher efficacy to produce interleukin-4 and interferon-γ and proliferation in response to MPV in comparison with the control. The prophylactic co-immunization regime caused significant breast tumors' growth inhibition, tumors' weight decrease, inhibition of metastasis formation, and enlarging tumor-bearing mice survival time, without any considerable side effects. Taking together, this cancer vaccine can evoke strong immune response against breast tumor and inhibits its growth and metastasis.

Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

Daily caloric restriction limits tumor growth more effectively than caloric cycling regardless of dietary composition.

Cancer incidence increases with age and is a leading cause of death. Caloric restriction (CR) confers benefits on health and survival and delays cancer. However, due to CR's stringency, dietary alternatives offering the same cancer protection have become increasingly attractive. Short cycles of a plant-based diet designed to mimic fasting (FMD) are protective against tumorigenesis without the chronic restriction of calories. Yet, it is unclear whether the fasting time, level of dietary restriction, or nutrient composition is the primary driver behind cancer protection. Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR.

The membrane-linked adaptor FRS2ß fashions a cytokine-rich inflammatory microenvironment that promotes breast cancer carcinogenesis.

Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2ß, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2ß deficiency in mouse mammary tumor virus (MMTV)-ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2ß-deficient premalignant tissues. We found that colocalization of FRS2ß and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor-κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB-induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2ß contain more stroma. The elucidation of the FRS2ß-NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

Abscopal Effect of Radiotherapy Enhanced with Immune Checkpoint Inhibitors of Triple Negative Breast Cancer in 4T1 Mammary Carcinoma Model.

Local radiotherapy (RT) is important to manage metastatic triple-negative breast cancer (TNBC). Although RT primarily reduces cancer cells locally, this control can be enhanced by triggering the immune system via immunotherapy. RT and immunotherapy may lead to an improved systemic effect, known as the abscopal effect. Here, we analyzed the antitumor effect of combination therapy using RT with an anti-programmed cell death-1 (PD-1) antibody in primary tumors, using poorly immunogenic metastatic mouse mammary carcinoma 4T1 model. Mice were injected subcutaneously into both flanks with 4T1 cells, and treatment was initiated 12 days later. Mice were randomly assigned to three treatment groups: (1) control (no treatment with RT or immune checkpoint inhibitor (ICI)), (2) RT alone, and (3) RT+ICI. The same RT dose was prescribed in both RT-alone and RT+ICI groups as 10Gy/fx in two fractions and delivered to only one of the two tumor burdens injected at both sides of flanks. In the RT+ICI group, 200 µg fixed dose of PD-1 antibody was intraperitoneally administered concurrently with RT. The RT and ICI combination markedly reduced tumor cell growth not only in the irradiated site but also in non-irradiated sites, a typical characteristic of the abscopal effect. This was observed only in radiation-sensitive cancer cells. Lung metastasis development was lower in RT-irradiated groups (RT-only and RT+ICI groups) than in the non-irradiated group, regardless of the radiation sensitivity of tumor cells. However, there was no additive effect of ICI on RT to control lung metastasis, as was already known regarding the abscopal effect. The combination of local RT with anti-PD-1 blockade could be a promising treatment strategy against metastatic TNBC. Further research is required to integrate our results into a clinical setting.

Effect of immune modulation on the skeletal muscle mitochondrial exercise response: An exploratory study in mice with cancer.

Cancer causes mitochondrial alterations in skeletal muscle, which may progress to muscle wasting and, ultimately, to cancer cachexia. Understanding how exercise adaptations are altered by cancer and cancer treatment is important for the effective design of exercise interventions aimed at improving cancer outcomes. We conducted an exploratory study to investigate how tumor burden and cancer immunotherapy treatment (anti-PD-1) modify the skeletal muscle mitochondrial response to exercise training in mice with transplantable tumors (B16-F10 melanoma and EO771 breast cancer). Mice remained sedentary or were provided with running wheels for ~19 days immediately following tumor implant while receiving no treatment (Untreated), isotype control antibody (IgG2a) or anti-PD-1. Exercise and anti-PD-1 did not alter the growth rate of either tumor type, either alone or in combination therapy. Untreated mice with B16-F10 tumors showed increases in most measured markers of skeletal muscle mitochondrial content following exercise training, as did anti-PD-1-treated mice, suggesting increased mitochondrial content following exercise training in these groups. However, mice with B16-F10 tumors receiving the isotype control antibody did not exhibit increased skeletal muscle mitochondrial content following exercise. In untreated mice with EO771 tumors, only citrate synthase activity and complex IV activity were increased following exercise. In contrast, IgG2a and anti-PD-1-treated groups both showed robust increases in most measured markers following exercise. These results indicate that in mice with B16-F10 tumors, IgG2a administration prevents exercise adaptation of skeletal muscle mitochondria, but adaptation remains intact in mice receiving anti-PD-1. In mice with EO771 tumors, both IgG2a and anti-PD-1-treated mice show robust skeletal muscle mitochondrial exercise responses, while untreated mice do not. Taken together, we postulate that immune modulation may enhance skeletal muscle mitochondrial response to exercise in tumor-bearing mice, and suggest this as an exciting new avenue for future research in exercise oncology.

2-Hexyl-4-Pentylenic Acid (HPTA) Stimulates the Radiotherapy-induced Abscopal Effect on Distal Tumor through Polarization of Tumor-associated Macrophages.

OBJECTIVE: The aim of this study was to explore the effects of 2-hexyl-4-pentylenic acid (HPTA) in combination with radiotherapy (RT) on distant unirradiated breast tumors. METHODS: Using a rat model of chemical carcinogen (7,12-dimethylbenz[a]anthracene,DMBA)-induced breast cancer, tumor volume was monitored and treatment response was evaluated by performing HE staining, immunohistochemistry, immunofluorescence, qRT-PCR, and western blot analyses. RESULTS: The results demonstrated that HPTA in combination with RT significantly delayed the growth of distant, unirradiated breast tumors. The mechanism of action included tumor-associated macrophage (TAM) infiltration into distant tumor tissues, M1 polarization, and inhibition of tumor angiogenesis by IFN-γ. CONCLUSION: The results suggest that the combination of HPTA with RT has an abscopal effect on distant tumors via M1-polarized TAMs, and HPTA may be considered as a new therapeutic for amplifying the efficacy of local RT for non-targeted breast tumors.The graphical abstract was available in the web of www.besjournal.com.

The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation.

During tumorigenesis, tumors must evolve to evade the immune system and do so by disrupting the genes involved in antigen processing and presentation or up-regulating inhibitory immune checkpoint genes. We performed in vivo CRISPR screens in syngeneic mouse tumor models to examine requirements for tumorigenesis both with and without adaptive immune selective pressure. In each tumor type tested, we found a marked enrichment for the loss of tumor suppressor genes (TSGs) in the presence of an adaptive immune system relative to immunocompromised mice. Nearly one-third of TSGs showed preferential enrichment, often in a cancer- and tissue-specific manner. These results suggest that clonal selection of recurrent mutations found in cancer is driven largely by the tumor's requirement to avoid the adaptive immune system.

Dietary Fats High in Linoleic Acids Impair Antitumor T-cell Responses by Inducing E-FABP-Mediated Mitochondrial Dysfunction.

The most recent American Dietary Guidelines (2020-2025) recommend shifting dietary fats from solid saturated fats to unsaturated oils. Dietary oils contain different compositions of unsaturated fatty acids (UFA). Oleic acid (OA) and linoleic acid (LA) are the most common UFA in dietary oils. How individual UFA in oils regulate immune cell function and cancer risk remains unclear. Here we demonstrated that high-fat diets (HFD) rich either in OA or LA induced a similar degree of murine obesity, but the LA-rich HFD specifically promoted mammary tumor growth. LA impaired antitumor T-cell responses by promoting naïve T-cell apoptosis and inhibiting TNFα production. While exogenous OA and LA were taken up by T cells with similar efficacy, only LA induced significant mitochondrial reactive oxygen species production and lipid peroxidation. Importantly, naïve T cells predominantly expressed epidermal fatty acid binding protein (E-FABP), which is central in facilitating LA mitochondrial transport and cardiolipin incorporation. Genetic depletion of E-FABP rescued LA-impaired T-cell responses and suppressed LA-rich HFD-associated mammary tumor growth. Collectively, these data suggest that dietary oils high in LA promote mammary tumors by inducing E-FABP-mediated T-cell dysfunction. SIGNIFICANCE: These findings suggest that modulation of dietary oil composition and inhibition of E-FABP activity may represent novel strategies to enhance T-cell function in the prevention and treatment of obesity-associated cancers.

Comparing syngeneic and autochthonous models of breast cancer to identify tumor immune components that correlate with response to immunotherapy in breast cancer.

BACKGROUND: The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic. METHODS: Three versions of a common syngeneic model derived from the MMTV-PyMT autochthonous model were generated by inoculating 1E6, 1E5, or 1E4 cells derived from the MMTV-PyMT mouse into wildtype recipient mice. To elucidate how tumor latency and TME heterogeneity contribute to ICB resistance, comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) was performed. Subsequently, response to ICB was tested. These procedures were repeated using the EMT6 breast cancer model. RESULTS: The 3 syngeneic versions of the MMTV-PyMT model had vastly different TMEs that correlated to ICB response. The number of cells used to generate syngeneic tumors significantly influenced tumor latency, infiltrating leukocyte populations, and response to ICB. These results were confirmed using the EMT6 breast cancer model. Compared to the MMTV-PyMT autochthonous model, all 3 MMTV-PyMT syngeneic models had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and higher proportions of PD-L1-positive myeloid cells, whereas the MMTV-PyMT autochthonous model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy, but PD-L1expression on tumor cells or PD-1 expression of T cells did not. CONCLUSIONS: These studies reveal that tumor cell number correlates with tumor latency, TME, and response to ICB. ICB-sensitive and resistant syngeneic breast cancer models were identified, in which the 1E4 syngeneic model was most resistant to ICB. Given the lack of benefit from ICB in breast cancer, identifying robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.

Antibody Repertoire Analysis of Tumor-Infiltrating B Cells Reveals Distinct Signatures and Distributions Across Tissues.

The role of B cells in the tumor microenvironment (TME) has largely been under investigated, and data regarding the antibody repertoire encoded by B cells in the TME and the adjacent lymphoid organs are scarce. Here, we utilized B cell receptor high-throughput sequencing (BCR-Seq) to profile the antibody repertoire signature of tumor-infiltrating lymphocyte B cells (TIL-Bs) in comparison to B cells from three anatomic compartments in a mouse model of triple-negative breast cancer. We found that TIL-Bs exhibit distinct antibody repertoire measures, including high clonal polarization and elevated somatic hypermutation rates, suggesting a local antigen-driven B-cell response. Importantly, TIL-Bs were highly mutated but non-class switched, suggesting that class-switch recombination may be inhibited in the TME. Tracing the distribution of TIL-B clones across various compartments indicated that they migrate to and from the TME. The data thus suggests that antibody repertoire signatures can serve as indicators for identifying tumor-reactive B cells.

PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8+T cells and promoting fatty acid metabolism.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) is frequently hyperactivated in cancer and plays important roles in both malignant and immune cells. The effect of PI3Kα inhibitors on the tumor microenvironment (TME) remains largely unknown. Here, we investigated the modulation of the TME by a clinical PI3Kα-specific inhibitor CYH33. METHODS: The activity of CYH33 against a panel of murine tumors in the immune-competent context or athymic mice was detected. Single-cell RNA sequencing and multi-parameter flow cytometry were performed to determine the immune profiling of TME. The effect of CYH33 on immune cells was conducted with primary murine cells. RESULTS: CYH33 exhibited more potent antitumor activity in immune-competent context. CYH33 enhanced the infiltration and activation of CD8+T and CD4+T cells, while attenuating M2-like macrophages and regulatory CD4+T cells. Increase in memory T cells was confirmed by the induction of long-term immune memory on CYH33 treatment. Mechanistically, CYH33 relieved the suppressed expansion of CD8+T cells via preferential polarization of the macrophages to the M1 phenotype. CYH33 promoted fatty acid (FA) metabolism in the TME, while FA enhanced the activity of CD8+T cells in vitro. The combination of CYH33 with the FA synthase (FASN) inhibitor C75 synergistically inhibited tumor growth with enhanced host immunity. CONCLUSIONS: CYH33 induces immune activation and synergizes with FASN inhibitor to further promote the antitumor immunity, which gains novel insights into how PI3K inhibitors exert their activity by modulating TME and provides a rationale for the concurrent targeting of PI3K and FASN in breast cancer treatment.